Abstract:
Objective This study aims to construct an ultra-large naive phage display nanobody library for screening antibody,for the diagnosis and treatment of infectious diseases.
Methods Total RNA was extracted from the spleen of one healthy alpaca and the peripheral blood of 100 healthy alpacas,then reversely transcribed into cDNA. The variable regions of the heavy-chain antibodies (VHH) were amplified by two rounds of polymerase chain reaction (PCR).The purified amplified products were ligated with the pSCD-2 vector,and then transformed into electrocompetent Escherichia coli TG1 cells to construct a phage display nanobody library.The positive transformation insertion rates,nanobody sequences,and contamination of antibody library were deteced and analyzed.
Results One naive nanobody library were successfully constructed, with a cumulative capacity of library reaching 1.165×1012, and the titer of the phage display library exceeded 1013 CFU/mL.The positive transformation insertion rates of nanobody library was 98.33% (177/180). Of the 125 randomly chosen PCR-positive clones for sequencing,all nanobody sequences were unique. The length of the complementarity-determining region 3 of the nanobodies ranged from 6 to 26 amino acids, showing a normal distribution and significant sequence diversity.No virulent phage contamination was found in the bacterial and and phage display antibody libraries.
Conclusion This study successfully constructed an ultra-large naive nanobody library with good diversity,laying a foundation for screening valuable and specific nanobodies.
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